The invention provides a culture medium for hybrid tumor cell clone and for preparing monoclonal antibodies and a preparation method thereof; RPMT 1640 complete media are prepared by conventional method, and then mixed with cell-free culture supernatant of the cells with certain proportions, such as BHK-21, CRFK, Hela and the like; the volume ratio of the complete media to each cell supernatant is (7-1):1. The advantages of the invention are as follows: due to the adoption of the inventive culture medium for hybrid tumor cell clone, the link in which BALB/c mouse peritoneal macrophage is used as feeder cells in the conventional hybrid tumor cells clone process is completely left out, which greatly simplifies clone operations and obviously increases the growth speed of the cloned cells.